Function and Regulation of a DEAD-box protein in mRNA Export

项目摘要 mRNA输出是真核生物基因表达程序的一个基本组成部分， 与上游（例如转录）和下游（例如RNA监视和翻译）事件相关。 mRNA输出模型的发展和对输出在调控基因中作用的理解 表达（例如选择转录物的偏向输出）需要定量评估mRNA输出， 包括动力学和分子机制。追求这一目标是及时给予的知识， 积累和技术开发（例如RNA适体标记系统），对于了解如何 疾病是在这种细胞过程受到干扰的情况下产生的。需要考虑的一个关键因素是Dbp 5 p，一个 死亡盒蛋白（DBP）通过调节RNA-蛋白质相互作用在输出中起关键作用， mRNA通过核孔复合物（NPC）的定向转运。Dbp 5 p被激活（即增强 ATP酶活性），虽然这些调节剂的影响， 已经在体外研究（例如稳态动力学和结构生物学），动力学和热力学 这种调节的起源，以及ATP酶循环如何影响mRNA输出的空间和时间方面， 体内，仍未解决。因此，建立精确的mRNA输出模型的必要步骤是： 定量了解（1）Dbp 5 p的ATP利用率和（2）Dbp 5 p对mRNA输出的影响 体内动力学，包括这些事件如何与NPC调节因子相互作用。 具体地说，速率和平衡常数的知识决定Dbp 5 p ATP酶活性和相互作用 与Nup 159 p和Gle 1 p是必要的发展预测NPC运输模型，可以结合 通过测量体内mRNA输出动力学和效率。目标1重点方面的拟议研究工作 瞬态（前稳态）动力学和定量平衡方法和分析，以确定如何 Nup 159 p和Gle 1 p与Dbp 5 p相互作用并调节Dbp 5 p。目的2和3采用体内研究，使用单 粒子RNA成像和转录组学方法来解决mRNA输出的空间和时间方面， 包括Dbp 5 p、Gle 1 p和Nup 159 p在调节输出中的作用。为了实现这些目标，新 实验和分析方法容易适用于其他DBPs和RNA运输系统将是 开发完成拟议的活动将建立一个定量分子框架， 描述了调节因子如何调节Dbp 5 p ATP酶活性和mRNA转运， 基因表达、细胞生理学和疾病病理学的基础。 !

PROJECT SUMMARY mRNA export is a fundamental component of the gene expression program in eukaryotes, which is tightly linked to upstream (e.g. transcription) and downstream (e.g. RNA surveillance and translation) events. Development of mRNA export models and an understanding of the role export plays in regulated gene expression (e.g. biased export of select transcripts) requires a quantitative assessment of mRNA export that encompasses kinetics and molecular mechanism. Pursuing this goal is timely given the knowledge that has amassed and technology developed (e.g. RNA-aptamer tagging systems) and is vital to understanding how disease arises in the context of perturbations to this cellular process. A key factor to consider is Dbp5p, a DEAD-box protein (DBP) that plays a critical role in export by modulating RNA-protein interactions important to directional transport of mRNAs through nuclear pore complexes (NPCs). Dbp5p is activated (i.e. enhanced ATPase activity) by the NPC components Gle1p and Nup159p, and although the impact of these regulators have been studied in vitro (e.g. steady-state kinetics and structural biology), the kinetic and thermodynamic origins of this regulation, and how ATPase cycling influences spatial and temporal aspects of mRNA export in vivo, remain unresolved. Therefore, a necessary step towards accurate models of mRNA export are a quantitative understanding of (1) ATP utilization by Dbp5p and (2) the impact of Dbp5p on mRNA export dynamics in vivo, including how these events are coupled to interactions with NPC regulatory factors. Specifically, knowledge of the rate and equilibrium constants dictating Dbp5p ATPase activity and interactions with Nup159p and Gle1p is necessary for developing predictive NPC transport models that can be combined with measurements of mRNA export kinetics and efficiency in vivo. Proposed research efforts in Aim 1 focus on transient (pre-steady state) kinetic and quantitative equilibrium methods and analyses to determine how Nup159p and Gle1p interact with and regulate Dbp5p. Aims 2 and 3 employ in vivo studies using single particle RNA imaging and transcriptomic approaches to address spatial and temporal aspects of mRNA export, including the role of Dbp5p, Gle1p and Nup159p in modulating export. To accomplish these aims, new experimental and analysis methods readily applicable to other DBPs and RNA transport systems will be developed. Completion of the proposed activities will establish a quantitative molecular framework that describes how regulatory factors function to modulate Dbp5p ATPase activity and mRNA transport, which is fundamental to gene expression, cell physiology, and disease pathology. !